Direct Reprogramming of Adult Human Cardiac Fibroblasts into Induced Cardiomyocytes Using miRcombo

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) through microRNAs (miRNAs) is a new emerging strategy for myocardial regeneration after ischemic heart disease. Previous studies have reported that murine fibroblasts can be directly reprogrammed into iCMs by transient transfection with four miRNAs (miRs-1, 133, 208 and 499 – termed “miRcombo”). While advancement in the knowledge of direct cell reprogramming molecular mechanism is in progress, it is important to investigate if this strategy may be translated to humans. Recently, we demonstrated that miRcombo transfection is able to induce direct reprogramming of adult human cardiac fibroblasts (AHCFs) into iCMs. Although additional studies are needed to achieve iCM maturation, our early findings pave the way toward new therapeutic strategies for cardiac regeneration in humans. This chapter describes methods for inducing direct reprogramming of AHCFs into iCMs through miRcombo transient transfection, showing experiments to perform for assessing iCM generatio

Direct cell reprogramming as a new emerging strategy in cardiac regeneration

Myocardial infarction (MI) is the current leading cause of mortality in the industrialised world. It is due to the irreversible death of billions of cardiomyocytes, secondary to a condition of ischemia. This leads to the formation of a stiff fibrotic tissue, mainly populated by cardiac fibroblasts (CFs). Currently, the only available therapy addressing the irreversible loss of functional cardiomyocytes is heart transplantation. Different tissue engineering approaches and cell therapies are under investigation, aimed at recovering myocardial contractility. Main issues in these strategies are the poor grafting and survival ability of implanted cells as well as the limited endogenous regenerative potential of adult heart. A new strategy is now emerging based on direct reprogramming of CFs into induced cardiomyocytes (iCMs) using transcriptional factors and/ or microRNAs (miRNAs) (miR-combo) [2-4]. Proof of concepts results of in vitro and in vivo conversion of mouse CFs into iCMs have been published and in vitro direct reprogramming of human CFs has also been reported [1-3]. However, such strategy is still an immature approach: reprogramming efficiency is low and partially reprogrammed non-beating cardiomyocytes have been generally obtained. Recently, in vitro direct reprogramming efficiency of mouse CFs cultured in 3D fibrin hydrogels using miR-combo has resulted significantly increased compared to 2D culture systems [4]. Based on these preliminary results, in this work we studied the miR-combo mediated reprogramming efficiency of human dermal and cardiac fibroblasts cultured on hydrogel matrices, including fibrin, fibrin/laminin, fibrin/fibronectin and fibrin/cardiac biomatrix [5], by analysing cell morphology, cell viability, change in gene expression (PCR analysis) and presence of markers of trans-differentiation by immunohistochemistry. The 3D biomimetic hydrogels were able to increase reprogramming efficiency respect to 2D culture environment, both at a genetic and protein level, with an enhancement in the expression of cardiac genes and cardiac proteins such as cardiac troponin I and alpha sarcomeric actinin. [1] J.A. Batty et al. Eur. J. Heart Failure 2016; 18: 145 [2] T.M. Jayawardena et al. Circ. Res. 2012; 110: 1465-1473. [3] T.M. Jayawardena et al. Circ. Res. 2015; 116:418-24. [4] Y. Li et al. Scientific Reports 2016; 6: 38815. [5] C. Castaldo et al. Biomed Res Int. 2013; 2013: 352370. ERC-CoG 2017 BIORECAR project is acknowledge

Role of anti-cyclic citrullinated peptide antibodies in discriminating patients with rheumatoid arthritis from patients with chronic hepatitis C infection-associated polyarticular involvement

This study was performed to assess the utility of anti-cyclic citrullinated peptide (anti-CCP) antibodies in distinguishing between patients with rheumatoid arthritis (RA) and patients with polyarticular involvement associated with chronic hepatitis C virus (HCV) infection. Serum anti-CCP antibodies and rheumatoid factor (RF) were evaluated in 30 patients with RA, 8 patients with chronic HCV infection and associated articular involvement and 31 patients with chronic HCV infection without any joint involvement. In addition, we retrospectively analysed sera collected at the time of first visit in 10 patients originally presenting with symmetric polyarthritis and HCV and subsequently developing well-established RA. Anti-CCP antibodies and RF were detected by commercial second-generation anti-CCP2 enzyme-linked immunosorbent assay and immunonephelometry respectively. Anti-CCP antibodies were detected in 23 of 30 (76.6%) patients with RA but not in patients with chronic HCV infection irrespective of the presence of articular involvement. Conversely, RF was detected in 27 of 30 (90%) patients with RA, 3 of 8 (37.5%) patients with HCV-related arthropathy and 3 of 31 (9.7%) patients with HCV infection without joint involvement. Finally, anti-CCP antibodies were retrospectively detected in 6 of 10 (60%) patients with RA and HCV. This indicates that anti-CCP antibodies can be useful in discriminating patients with RA from patients with HCV-associated arthropathy

In vitro mechanical stimulation to reproduce the pathological hallmarks of human cardiac fibrosis on a beating chip and predict the efficacy of drugs and advanced therapies

Cardiac fibrosis is one of the main causes of heart failure, significantly contributing to mortality. The discovery and development of effective therapies able to heal fibrotic pathological symptoms thus remain of paramount importance. Micro-physiological systems (MPS) are recently introduced as promising platforms able to accelerate this finding. Here a 3D in vitro model of human cardiac fibrosis, named uScar, is developed by imposing a cyclic mechanical stimulation to human atrial cardiac fibroblasts (AHCFs) cultured in a 3D beating heart-on-chip and exploited to screen drugs and advanced therapeutics. The sole provision of a cyclic 10% uniaxial strain at 1 Hz to the microtissues is sufficient to trigger fibrotic traits, inducing a consistent fibroblast-to-myofibroblast transition and an enhanced expression and production of extracellular matrix (ECM) proteins. Standard of care anti-fibrotic drugs (i.e., Pirfenidone and Tranilast) are confirmed to be efficient in preventing the onset of fibrotic traits in uScar. Conversely, the mechanical stimulation applied to the microtissues limit the ability of a miRNA therapy to directly reprogram fibroblasts into cardiomyocytes (CMs), despite its proved efficacy in 2D models. Such results demonstrate the importance of incorporating in vivo-like stimulations to generate more representative 3D in vitro models able to predict the efficacy of therapies in patients

Lipoplexes for effective in vitro delivery of microRNAs to adult human cardiac fibroblasts for perspective direct cardiac cell reprogramming

Design of nanocarriers for efficient miRNA delivery can significantly improve miRNA-based therapies. Lipoplexes based on helper lipid, dioleoyl phosphatidylethanolamine (DOPE) and cationic lipid [2-(2,3-didodecyloxypropyl)-hydroxyethyl] ammonium bromide (DE) were formulated to efficiently deliver miR-1 or a combination of four microRNAs (miRcombo) to adult human cardiac fibroblasts (AHCFs). Lipoplexes with amino-to-phosphate groups ratio of 3 (N/P 3) showed nanometric hydrodynamic size (372 nm), positive Z-potential (40 mV) and high stability under storage conditions. Compared to commercial Dharma FECT1 (DF), DE-DOPE/miRNA lipoplexes showed superior miRNA loading efficiency (99 % vs. 64 %), and faster miRNA release (99 % vs. 82 % at 48 h). DE-DOPE/miR-1 lipoplexes showed superior viability (80-100 % vs. 50 %) in AHCFs, a 2-fold higher miR-1 expression and Twinfilin-1 (TWF-1) mRNA down regulation. DE-DOPE/ miRcombo lipoplexes significantly enhanced AHCFs reprogramming into induced cardiomyocytes (iCMs), as shown by increased expression of CM markers compared to DF/miRcombo. (C) 2022 The Authors. Published by Elsevier Inc

The new integrated aeromagnetic map of the Phlegrean Fields volcano and surrounding areas

In this paper we present and analyze the new detailed aeromagnetic data set resulting from a recent survey car- ried out in the Phlegrean Fields volcanic area. The survey was aimed at gaining new insight into the vol- canological characteristics of the region north of Phlegrean Fields (Parete-Villa Literno area) where remarkable thickness of volcanic/sub-volcanic rocks were found in wells. Measurement of total magnetic field was per- formed on two different flight levels, 70 m and 400 m above the ground surface, along flight lines spaced 400 m apart. Both aeromagnetic maps show the noisy effect of linear anomalies evidently due to the presence of rail- way lines. To filter out these local anomalies a method based on discrete wavelet transform was used, allowing an accurate local filtering and leaving the rest of the field practically unchanged. The filtered data set was inte- grated with the existing Agip aeromagnetic map of the Phlegrean Fields, leading to a new aeromagnetic map of the whole Phlegrean volcanic area. The compilation of the pole reduced map and of the maps of the Analytic Signal and of the Horizontal Derivative of the integrated data set represents a first step for the interpretation of the maps in terms of geological structures of the whole Phlegrean volcanic district